Biochemical characterization and comparison of aspartylglucosaminidases secreted in venom of the parasitoid wasps <i>Asobara tabida</i> and <i>Leptopilina heterotoma</i>

<div><p>Aspartylglucosaminidase (AGA) is a low-abundance intracellular enzyme that plays a key role in the last stage of glycoproteins degradation, and whose deficiency leads to human aspartylglucosaminuria, a lysosomal storage disease. Surprisingly, high amounts of AGA-like proteins are secreted in the venom of two phylogenetically distant hymenopteran parasitoid wasp species, <i>Asobara tabida</i> (Braconidae) and <i>Leptopilina heterotoma</i> (Cynipidae). These venom AGAs have a similar domain organization as mammalian AGAs. They share with them key residues for autocatalysis and activity, and the mature α- and β-subunits also form an (αβ)₂ structure in solution. Interestingly, only one of these AGAs subunits (α for AtAGA and β for LhAGA) is glycosylated instead of the two subunits for lysosomal human AGA (hAGA), and these glycosylations are partially resistant to PGNase F treatment. The two venom AGAs are secreted as fully activated enzymes, they have a similar aspartylglucosaminidase activity and are both also efficient asparaginases. Once AGAs are injected into the larvae of the <i>Drosophila melanogaster</i> host, the asparaginase activity may play a role in modulating their physiology. Altogether, our data provide new elements for a better understanding of the secretion and the role of venom AGAs as virulence factors in the parasitoid wasps’ success.</p></div

Alignment of AGA sequences.

<p>Translated cDNA sequences of <i>A</i>. <i>tabida</i> (AtAGA; ACX94224) and <i>L</i>. <i>heterotoma</i> venom AGAs (LhAGA; KP888635) aligned with the sequence of human AGA (hAGA; P20933). Residues identical with hAGA are indicated by a dot. Signal peptide amino acids are in blue. Conserved residues important for structure or activity are highlighted in green, with an asterisk indicating the active-site threonine (hAGA T206). Important hAGA residues not conserved in AtAGA and/or LhAGA are highlighted in red. Cysteine residues are framed by a black box and those involved in disulfide bonds are connected with black solid lines. Potential N-glycosylation sites are in brown (hAGA N38 and N308, AtAGA N52 and N153, LhAGA N326). Residues important for hAGA phosphorylation are in purple (K177, Y178, K183 and K214).</p

FPLC purification profiles of AtAGA and LhAGA.

<p>A and D. FPLC profiles of <i>A</i>. <i>tabida</i> (A) and <i>L</i>. <i>heterotoma</i> (D) venom extracts at 280 nm. B and E. 12.5% SDS-PAGE analysis of each FPLC fraction for <i>A</i>. <i>tabida</i> (B) and <i>L</i>. <i>heterotoma</i> (E). Lane T, total venom extract (3 venom apparatus/well). Aspartylglucosaminidase activity measured with 20 μl of each FPLC fraction is overlay on gel pictures for AtAGA (B) and LhAGA (E). C and F. Detection of AGA on western-blots of 10 μl of each FPLC fraction of <i>A</i>. <i>tabida</i> (anti-P30 antibody, C) and <i>L</i>. <i>heterotoma</i> (anti-LhAGA, F). Only AGA positive fractions are shown.</p

Analysis of the native conformation of AtAGA and LhAGA.

<p>A. Non-reducing/reducing electrophoretic migration of <i>L</i>. <i>heterotoma</i> venom. After venom separation on a 12.5% non-reducing gel, the full lane was excised and run under reducing conditions (gel on the left). The only band that showed a migration shift (boxed) was excised, run under reducing conditions, and silver stained (Gel) or probed with the anti-LhAGA (WB) (lanes on the right). B. Glutaraldehyde cross-linking analysis of the oligomerization state of FPLC purified native AtAGA and LhAGA (% of glutaraldehyde on top of the lane). First lane: molecular weight markers.</p

Kinetic constants of AtAGA and LhAGA for AspAMC and L-asparagine.

<p>Kinetic constants of AtAGA and LhAGA for AspAMC and L-asparagine.</p

Homology modeling of AtAGA and LhAGA.

<p>A. Superposition of the active (αβ)₂ tetramer tertiary model structure of mature AtAGA (QMEAN score = 0.694; QMEAN Z-score = -0.83; colored in red) and LhAGA (QMEAN score = 0.725; QMEAN Z-score = -0.5; colored in green) with the solved structure of human AGA (hAGA; 1APY; colored in cyan). B. Spatial geometry of some of the key catalytic and binding residues of hAGA, AtAGA and LhAGA active sites.</p